During development of the nervous system, axons extend stepwise toward destinations via intermediate targets. Genetic analysis in Drosophila has identified the ventral midline of the nerve cord as an important intermediate target. Ventral midline cells attract axons of dorsally positioned neurons, but also secrete the axonal repellent, Slit. Despite the presence of Slit, commissural axons traverse the midline due to downregulation of their Slit receptors - members of the Robo family - by the Comm protein. While Slit-Robo signaling also regulates commissural axon projections in vertebrates, no vertebrate Comm homologue has been identified. The objective of this proposal is to demonstrate that Robo3 plays a functionally equivalent role to Comm in vertebrate axon guidance at the ventral midline. To do this I will analyze commissural axon pathfinding in the zebrafish twitch twice mutant. In the twitch twice hindbrain, axons which normally cross the midline fail to do so and instead project caudally along ipsilateral tracts. The mutation in twitch twice disrupts the Robo3 gene. Robo3 lacks conserved Robo family signaling motifs and may prevent signaling through other Robo proteins. I hypothesize that before crossing the midline, Robo3 silences Slit-Robo signaling, and is thus functionally equivalent to Comm. The experiments in this proposal will: First, test the hypothesis that Robo3 is selectively required in commissural neurons. Second, test the hypothesis that the molecular function of Robo3 is to disrupt Slit-Robo signaling. Third, define the role of Robo3 in axon pathfinding after midline crossing. The results of these experiments will reveal how commissural axon pathfinding is regulated in vertebrates.